Leaf respiratory CO2 is 13 C-enriched relative to leaf organic components in five species of C3 plants.

•  Here, we compared the carbon isotope ratios of leaf respiratory CO2 (δ13 CR ) and leaf organic components (soluble sugar, water soluble fraction, starch, protein and bulk organic matter) in five C3 plants grown in a glasshouse and inside Biosphere 2. One species, Populus deltoides, was grown under three different CO2 concentrations. •  The Keeling plot approach was applied to the leaf scale to measure leaf δ13 CR and these results were compared with the δ13 C of leaf organic components. •  In all cases, leaf respiratory CO2 was more 13 C-enriched than leaf organic components. The amount of 13 C enrichment displayed a significant species-specific pattern, but the effect of CO2 treatment was not significant on P. deltoides. •  In C3 plant leaves, 13 C-enriched respiratory CO2 appears widespread. Among currently hypothesized mechanisms contributing to this phenomenon, non-statistical carbon isotope distribution within the sugar substrates seems most likely. However, caution should be taken when attempting to predict the δ13 C of leaf respiratory CO2 at the ecosystem scale by upscaling the relationship between leaf δ13 CR and δ13 C of leaf organic components.